The present invention relates to an array of perfused bioreactors in a “multiwell plate” format, wherein each bioreactor of the array consists of a microscale matrix seeded with cells which forms a microtissue, multiple tissues, and/or cell aggregate and methods for using the array in high throughput assays, for example, for determining the effect of biological and/or chemical agents on the microscale tissue arrays, studying tissue-tissue interactions, or for detecting the presence of biological and/or chemical agents.
Tissue engineering has emerged as a scientific field which has the potential to aid in human therapy by producing anatomic tissues and organs for the purpose of reconstructive surgery and transplantation. It combines the scientific fields of materials science, cell and molecular biology, and medicine to yield new devices for replacement, repair, and reconstruction of tissues and structures within the body. Many approaches have been advocated over the last decade. One approach is to combine tissue specific cells with open porous polymer scaffolds which can then be implanted. Large numbers of cells can be added to the polymer device in cell culture and maintained by diffusion. After implantation, vascular ingrowth occurs, the cells remodel, and a new stable tissue is formed as the polymer degrades by hydrolysis.
A number of approaches have been described for fabricating tissue regeneration devices for either in vitro or in vivo growth of cells. Polymeric devices have been described for replacing organ function or providing structural support. Such methods have been reported by Vacanti, et al., Arch. Surg. 123:545-49 (1988); U.S. Pat. No. 4,060,081 to Yannas, et al.; U.S. Pat. No. 4,485,097 to Bell; and U.S. Pat. No. 4,520,821 to Schmidt, et al. In general, the methods used by Vacanti, et al., and Schmidt, et al., can be practiced by selecting and adapting existing polymer fiber compositions for implantation and seeding with cells, while the methods of Yannas and Bell produce very specific modified collagen sponge-like structures.
Tissue regeneration devices must be porous with interconnected pores to allow cell and tissue penetration, if the device is of any significant thickness. Factors such as pore size, shape, and tortuosity can all affect tissue ingrowth but are difficult to control using standard processing techniques. U.S. Pat. No. 5,518,680 to Cima & Cima describes the use of solid free form fabrication techniques, especially three dimensional printing of polymer powders, to form matrices which can be seeded with dissociated cells and implanted to form new structures. The advantages of the solid free form methods to construct specific structures from biocompatible synthetic or natural polymers, inorganic materials, or composites of inorganic materials with polymers, where the resulting structure has defined pore sizes, shapes and orientations, particularly different pore sizes and orientations within the same device, with more than one surface chemistry or texture at different specified sites within the device, is readily apparent. However, the devices still have a major limitation: ingrowth of new tissue to form blood vessels which sustain the implanted cells must occur at the right time relative to the increasing cell density within the matrix to sustain the implanted cells, and other tissues must not encapsulate or infiltrate the matrix to choke out or otherwise destroy the implanted cells.
PCT/US96/09344 to Massachusetts Institute of Technology and Childrens' Medical Center Corporation describes the use of solid free-form fabrication (SFF) methods to manufacture devices for allowing tissue regeneration and for seeding and implanting cells to form organ and structural components, which can additionally provide controlled release of bioactive agents, wherein the matrix is characterized by a network of lumens functionally equivalent to the naturally occurring vasculature of the tissue formed by the implanted cells, and which can be lined with endothelial cells and coupled to blood vessels or other ducts at the time of implantation to form a vascular or ductile network throughout the matrix.
None of this technology, however, provides a means to maintain the tissue in vitro, nor to use the tissue as a diagnostic or screening tool. Cells placed in typical in vitro culture generally lose at least some key differentiated physiological functions that they normally exhibit as part of organized tissues in the body. Thus, while cultured cells may be adequate for certain applications, for example, in detection of toxins and pathogens, they are certain to fail in other applications, for example, screening of drug which are metabolized by the tissues, or drugs which are cleared through interaction with a complex organ, not just a single isolated cell type. For example, no in vitro model of infection exists for hepatitis B virus (HBV) and hepatitis C virus (HCV), presumably because primary hepatocytes in typical culture situations rapidly stop expressing the cell surface receptors the viruses use to enter the cell. One can infer from this example of a known pathogen, which cannot currently be screened using cultured cells, that unknown pathogens (or toxins), which often utilize receptor-mediated uptake, could similarly elude detection in cultured cells. Similarly, drugs that must be bound by cell specific receptors to be taken up by the cells to be active, also cannot be tested in such systems. Xenobiotic metabolism, which is primarily carried out by a set of enzymes in the liver, is another function rapidly lost by cultured hepatocytes. Although the hepatic enzymes render most exogenous compounds less toxic, other molecules (as a common example, the pain-relieving drug acetaminophen) can actually become more toxic when metabolized by the liver. It is therefore critical to have a system for screening of drugs which can mimic in vivo conditions.
Currently, no in vitro models or animal models adequately capture the complex responses of human tissues to drugs and environmental agents. Furthermore, no in vitro model captures the complex biology of tumor cell interactions with adjacent normal tissues. Each year, many new drugs fail in early clinical trials due to unanticipated toxicity, especially liver toxicity, or due to failure to account for liver metabolism of anti-tumor agents. Liver cells rapidly lose liver-specific functions when placed in culture. Thus assessing long-term toxicity of drugs on human liver tissue in vitro is not possible. Further, sources of human liver cells are scarce, and are not able to meet the demand for cell and tissue-based assays in the pharmaceutical industry. Further, the ability to culture human tissue provides the opportunity to create models of single-cell metastasis and the very first stages of tumor growth, providing a means to culture difficult-to-culture cancer, to predict the propensity of a given tumor to metastasize and grow, to study tumor biology, and to test the efficacy of anti-cancer compounds.
The current state-of-the art 2D and 3D culture methods do not enable perfusion through the tissue mass in a manner that replicates physiological flow.
U.S. Pat. No. 6,197,575 to Griffith, et al., describes a micromatrix and a perfusion assembly suitable for seeding, attachment, and culture of complex hierarchical tissue or organ structures.
It is therefore an object of the present invention to provide an apparatus for in vitro analyses that effectively model disease in tissue and/or organs but which does not require tissue or organs.